Cigarette Smoke Effects on Lung Cells

BEAS-2B cells (SV40-immortalized human airway bronchial epithelial cell line) and A549 cells (human lung adenocarcinoma epithelial cell line) were purchased from the American Culture of Tissue Collection and grown in complete growth medium (RPMI 1640 and DMEM supplemented with heat-inactivated 10% FBS and 1% L-glutamine, respectively) at 37^oC/5% CO₂. Before use, cells were starved in minimum medium (RPMI 1640 or DMEM supplemented with 1% FBS and 1% L-glutamine), and cell culture supernatants were harvested at different time points. To eliminate the contamination of supernatant by free-floating cells, the supernatant was centrifuged and the upper half of the medium was taken as the sample.
Human primary bronchial epithelial cells obtained from three subjects without COPD and three subjects with COPD were cultured as monolayers in LHC-9 media (Invitrogen) on collagen (1% w/v)-coated plates as previously reported.⁴⁰ (link) Cells were extracted from lung tissue of patients undergoing lung resection at the Royal Brompton Hospital. All subjects gave informed written consent and the study was approved by the National Research Ethics Service London-Chelsea Research Ethics committee, study No. 09/H0801/85. All cells were serum starved 16 hours before stimulation. Cells were stimulated with 3% cigarette-smoke-conditioned media prepared as previously reported.⁴¹ (link)